Diffuse large B cell lymphoma (DLBCL) is molecularly heterogeneous disease that is not yet completely understood. Following standard immunochemotherapy, 40% of patients succumb to their disease, signifying the need to identify new targets. Heat shock protein 90 (Hsp90), an emerging therapeutic target for cancer, is a chaperone protein that assists the correct folding of signaling mediators involved in cell growth, proliferation and survival. Tumor cells are enriched for a fraction of Hsp90 found in multi-chaperone complexes (tumor-enriched Hsp90, teHsp90) that have high affinity for Hsp90 inhibitors. teHsp90 client proteins are depleted when tumor cells are exposed to teHsp90 inhibition (teHsp90i). We have shown that PUH71, a novel, highly teHsp90 selective inhibitor, has potent activity in different DLBCL cell lines and xenografts. By immobilizing PUH71 on a solid support, teHsp90 complexes can be chemically precipitated (CP), allowing us to identify teHsp90 client proteins. Using this method, we validated the discovery that BCL6, the main oncoprotein in BCLs, is a teHsp90 substrate protein. We have also shown that combined inhibition of teHsp90 and its client, BCL6, synergizes to kill DLBCLs. To better understand lymphoma-specific teHsp90 mechanisms of action and to identify new targets for mono- or combination therapy, we performed PUH71 CP proteomics in DLBCL cell lines. Many components of the B cell receptor (BCR) pathway were identified as clients of teHsp90. This pathway has been implicated in lymphomagenesis and DLBCL survival. We hypothesize that teHsp90 chaperones BCR signalosome formation and activity and that combined inhibition of teHsp90 and the BCR pathway will synergize to kill DLBCLs. To test this hypothesis we will assess PUH71 treated cells for changes in BCR signalosome components and downstream signaling at the mRNA (qPCR, mRNA-seq), protein (IB), and phospho-protein (FC) levels. Total internal reflection fluorescence microscopy (TIRFM) will be used to depict the effect of teHsp90i on BCR signalosome formation and stability. To assess effects of teHsp90i on BCR signaling, we will evaluate changes in calcium release, CARD11-Bcl10-MALT1 complex formation (IP, WB), and transcriptional activity (qPCR). We will treat DLBCL cell lines with BCR pathway inhibitors alone and in combination with teHsp90i and evaluate for additive or synergistic effect using isobologram analysis. Effective combination treatments will be translated to in vivo xenograft models and ex vivo patient samples. The proposed experiments will characterize a new function for teHsp90 and provide the rationale for translating effective combination treatments to patients in the clinic.